Validation of equivalent bacterial load of P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i> infected Balb/c mice.

<p>Skin samples from adjacent to the inoculation site of Balb/c mice infected with 10⁵ P_<i>flaB</i><i>-luc</i> or P_<i>ospC</i><i>-luc</i> on day 10 (A) or day 21 (B) following inoculation were harvested for qPCR analysis of borrelial genomes (<i>recA</i>) per copies of 10⁶ β-actin. Horizontal bars denotes average copies of <i>recA</i> per 10⁶ β-actin and error bars represent standard error. Statistical analysis using the Mann-Whitney test indicated a lack of significance between the P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i> at day 10 and 21 post-infection with <i>p</i>-values of 0.2222 and 0.9444, respectively.</p

<i>In Vivo</i> Imaging Demonstrates That <i>Borrelia burgdorferi ospC</i> Is Uniquely Expressed Temporally and Spatially throughout Experimental Infection

<div><p><i>Borrelia burgdorferi</i> is a spirochetal bacterium transmitted by the <i>Ixodes</i> tick that causes Lyme disease in humans due to its ability to evade the host immune response and disseminate to multiple immunoprotective tissues. The pathogen undergoes dynamic genetic alterations important for adaptation from the tick vector to the mammalian host, but little is known regarding the changes at the transcriptional level within the distal tissues they colonize. In this study, <i>B</i>. <i>burgdorferi</i> infection and gene expression of the essential virulence determinant <i>ospC</i> was quantitatively monitored in a spatial and temporal manner utilizing reporter bioluminescent borrelial strains with <i>in vivo</i> and <i>ex vivo</i> imaging. Although expressed from a shuttle vector, the P_<i>ospC</i>-<i>luc</i> construct exhibited a similar expression pattern relative to native <i>ospC</i>. Bacterial burden in skin, inguinal lymph node, heart, bladder and tibiotarsal joint varied between tissues and fluctuated over the course of infection possibly in response to unique cues of each microenvironment. Expression of <i>ospC</i>, when normalized for changes in bacterial load, presented unique profiles in murine tissues at different time points. The inguinal lymph node was infected with a significant <i>B</i>. <i>burgdorferi</i> burden, but showed minimal <i>ospC</i> expression. <i>B</i>. <i>burgdorferi</i> infected skin and heart induced expression of <i>ospC</i> early during infection while the bladder and tibiotarsal joint continued to display P_<i>ospC</i> driven luminescence throughout the 21 day time course. Localized skin borrelial burden increased dramatically in the first 96 hours following inoculation, which was not paralleled with an increase in <i>ospC</i> expression, despite the requirement of <i>ospC</i> for dermal colonization. Quantitation of bioluminescence representing <i>ospC</i> expression in individual tissues was validated by qRT-PCR of the native <i>ospC</i> transcript. Taken together, the temporal regulation of <i>ospC</i> expression in distal tissues suggests a role for this virulence determinant beyond early infection.</p></div

Correlation of <i>ospC</i> radiance and quantitation measure of <i>ospC</i> transcript in mammalian tissues.

<p>P_<i>ospC</i><i>-luc</i> infected tissues from day 10 and 21 post-infection were evaluated for radiance (p/sec/cm²/sr) relative to total <i>ospC</i> transcript for the whole tissue sample. Skin (A), heart (B), bladder (C), and tibiotarsal joint (D) had <i>r</i> values of 0.944, 0.961, 0.854, and 0.812, respectively.</p

Characterization of a bioluminescent <i>B</i>. <i>burgdorferi</i> P_<i>ospC</i> reporter strain.

<p>The response of borrelial <i>ospC</i> reporter strain to pH was assessed to determine the validity of P_<i>ospC</i><i>-luc</i> relative to native OspC production. P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc B</i>. <i>burgdorferi</i> strains were grown at pH 7 and pH 8 to mid-log phase to assess <i>in vitro</i> luminescence assay and protein production via Western blot analysis. (A) P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i> cultures were serial diluted from 10⁶ to 10 cells, treated with D-luciferin, and luminescence was measured in photons/sec. P_<i>flaB</i><i>-luc</i> pH 7 (red squares) and P_<i>flaB</i><i>-luc</i> pH 8 (black circles) did not differ in bioluminescence. P_<i>ospC</i><i>-luc</i> pH 7 (blue squares) induces greater luminescence relative to P_<i>ospC</i><i>-luc</i> pH 8 (green circles). Values represent three independent cultures that were normalized to background and averaged. Error bars represent standard error. (B) Differential protein production of Luc in P_<i>ospC</i><i>-luc</i> reporter strain in response to pH reflects changes observed for the native OspC in P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i>. Cell lysates of P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i> at pH 7 or pH 8 were immunoblotted and probed with anti-sera against OspC, FFluc and FlaB that served as a loading control.</p

Primers used in this study.

<p>Primers used in this study.</p

Evaluation of <i>ospC</i> expression as represented by P_<i>ospC</i><i>-luc</i> expressing <i>B</i>. <i>burgdorferi</i> during early infection.

<p>Balb/c mice infected by ventral intradermal injection with 10⁵ P_<i>ospC</i><i>-luc</i> and P_<i>flaB</i><i>-luc B</i>. <i>burgdorferi</i> for the quantitation of <i>ospC</i> relative to bacterial load during the 96 hours following needle inoculation. (A) Mice were treated with D-luciferin and imaged 2, 8, 24, 48, 72, and 96 hours with the exception of one no D-luciferin background control at each time point. Ten minute exposures were used to obtain images and were normalized to radiance range of 6.95x10³-1.83x10⁵ p/sec/cm²/sr. (B) Quantification of bioluminescence was determined from 1 minute exposure images. Values represent flux (photon/sec) normalized to background and the averaged value from four mice treated with D-luciferin. P_<i>ospC</i><i>-luc</i> is represented by red squares and P_<i>flaB</i><i>-luc</i> is represented by blue circles. Error bars represent standard error. An asterisk represents <i>p</i> < 0.05, indicating significant differences in bioluminescence between P_<i>ospC</i><i>-luc</i> and P_<i>flaB</i><i>-luc</i> containing <i>B</i>. <i>burgdorferi</i>. There was no significant difference in P_<i>ospC</i><i>-luc</i> radiance during the first 96 hours. One-way ANOVA of P_<i>flaB</i><i>-luc</i> radiance had a of <i>p</i>-value < 0.0001 indicating a statistically significant change in luminescence.</p

Strains and Plasmids used in this study.

<p>Strains and Plasmids used in this study.</p

Quantitation of P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i> expressing <i>B</i>. <i>burgdorferi</i> using bioluminescent readout.

<p>Five Balb/c mice were infected with 10⁵ P_<i>flaB</i><i>-luc</i> or P_<i>ospC</i><i>-luc</i> containing <i>B</i>. <i>burgdorferi</i> and 4 mice treated with D-luciferin for imaging 0, 4, 7, 10, 14, and 21 days post-inoculation for bioluminescent imaging. (A) Quantitation of 1 minute exposures was performed. At all time points the whole mouse was measured to obtain a measurement in photons/sec, representing total flux. Bioluminescence from the 4 mice treated with D-luciferin was normalized by subtracting the measurement from the no D-luciferin control and averaged. Blue circles represent P_<i>flaB</i><i>-luc</i> and red squares P_<i>ospC</i><i>-luc</i>. Error bars represent standard error. One-way ANOVA analysis resulted in statistical significance for P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i> with a <i>p</i>-value of 0.0132 and 0.0262, respectively. (B) To assess the expression of <i>ospC</i> independent of changes in borrelial load, the ratio differential of P_<i>ospC</i><i>-luc</i> and P_<i>flaB</i><i>-luc</i> was calculated and represented on the y-axis. Permutation analyses comparing time points to each other found statistically significant differences (<i>p</i> < 0.05) between all comparisons, except between day 4 and day 21.</p

Temporal and spatial expression of <i>B</i>. <i>burgdorferi</i> containing P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i> in murine tissues.

<p>Skin, inguinal lymph node, heart, bladder, and tibiotarsal joint, from P_<i>flaB</i><i>-luc</i> or P_<i>ospC</i><i>-luc</i> infected Balb/c mice were quantitatively assessed for bioluminescent emission at 4, 7, 10, 14 and 21 days post-infection. Four of the five mice were treated with a double bolus of D-luciferin and the remaining mouse served as a background control for normalization of <i>ex vivo</i> tissues. The—represents the no luciferin control and + designates the tissues treated with D-luciferin. Tissues were evaluated for bacterial load or <i>ospC</i> expression as represented by P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i>, respectively. Normalization to subtract background was performed per strain for all time points displayed in the color spectrum position under the images. P_<i>flaB</i><i>-luc</i> and P_<i>ospC</i><i>-luc</i> images are set on individual scales to display the full spectrum of bioluminescence over the experimental time course. Measurable radiance above background is detectable on all days evaluated in all tissues with the exception of P_<i>ospC</i><i>-luc</i> infected inguinal lymph node that emits minimal luminescence. Graphs for each tissue display the ratio of P_<i>ospC</i><i>-luc/</i>P_<i>flaB</i><i>-luc</i> to depict the expression of <i>ospC</i> as measured by P_<i>ospC</i><i>-luc</i> relative to bacterial load (scored by P_<i>flaB</i><i>-luc</i>) for a given time point. The P_<i>ospC</i><i>-luc/</i>P_<i>flaB</i><i>-luc</i> ratio underwent permutation analyses comparing all time points to determine statistical significance. (A) The radiance range of skin at the site of inoculation for both strains is 4.9e3-1.83e5 p/sec/cm²/sr. All comparisons have a <i>p</i>-value < 0.05. (B) Heart radiance for P_<i>flaB</i><i>-luc</i> is 4.3e2-1.1e4 and P_<i>ospC</i><i>-luc</i> is 3.92e2-3.9e3 p/sec/cm²/sr. Two comparisons, day 4 versus day 21 and day 7 versus day 14, were not statistically significant. Remaining comparisons were significantly different (<i>p</i>-value<0.05). (C) Inguinal lymph node radiance for P_<i>flaB</i><i>-luc</i> is 1.1e3-1.1e4 and P_<i>ospC</i><i>-luc</i> is 8.32e2-1.5e3 p/sec/cm²/sr. Comparisons were statistically significant with <i>p</i>-values of 0.0160 or less, except day 10 versus day 21. (D) Bladder radiance for P_<i>flaB</i><i>-luc</i> is 1.1e3-5e4 and P_<i>ospC</i><i>-luc</i> is 3.78e2-1.1e4 p/sec/cm²/sr. There is statistical difference between early time points (day 4, 7, & 10) and late time points (day 14 & 21) with <i>p</i>-values no greater than 0.0305. (E) Tibiotarsal joint radiance for P_<i>flaB</i><i>-luc</i> is 1.15e3-1.15e5 and P_<i>ospC</i><i>-luc</i> is 4.95e2-1.1e4 p/sec/cm²/sr. All comparisons were statistically different (<i>p</i>-value < 0.05), except for day 4 versus day 21.</p

Frequency ratios of individual Tn mutants with insertions in putative ROS and RNS resistance genes.

<p>We prioritized genes for further study if they had overall frequency ratios <0.33 in both replicates after exposure to DEA/NO (A), TBHP (B), or H₂O₂ (C). The frequency ratios of individual Tn mutants with insertions in these genes are shown for both replicate 1 (black circles) and replicate 2 (gray circles). The median is indicated with a bar. Genes that were selected for further analysis are shown in blue.</p